Lyme disease (Lyme borreliosis) is the most common tick-borne infectious disease in North America and Europe, and has been found in Russia, Japan, China and Australia. Lyme disease begins at the site of a tick bite, producing a primary infection with spread of the organism to secondary sites occurring during the course of infection. The causative bacterial agent of this disease is the spirochete Borrelia burgdorferi, which was first isolated and cultivated in 1982 (Burgdorferi, W. A. et al., Science 216: 1317-1319 (1982); Steere, A. R. et al., N. Engl. J. Med. 308: 733-740 (1983)). With that discovery, a wide array of clinical syndromes, described in both the European and American literature since the early 20th century, could be attributed to infection by B. burgdorferi (Afzelius, A., Acta Derm. Venereol. 2: 120-125 (1921); Bannwarth, A., Arch. Psychiatr. Nervenkrankh. 117: 161-185 (1944); Garin, C. and A. Bujadouz, J. Med. Lyon 71: 765-767 (1922); Herxheimer, K. and K. Hartmann, Arch. Dermatol. Syphilol. 61: 57-76, 255-300 (1902)).
Three pathogenic genospecies of Borrelia, B. burgdorferi sensu stricto (B. burgdorferi or B.b.s.s.), B. afzelii and B. garinii have been described (Baranton, G., et al., Int. J. Syst. Bacteriol. 42:378-383 (1992)). These are members of a species complex, B. burgdorferi sensu lato, which consists of at least 10 different genospecies (Piken, R. N., et al., J. Invest. Dermatol., 110:211-214 (1998); Postic, D., et al., Int. J. Syst. Bacteriol. 44:743-752 (1994); Valsangiacomo, C. T., et al., Int. J. Syst. Bacteriol. 47:1-10 (1997)). The three genospecies, B. burgdorferi sensu stricto, B. afzelii and B. garinii, all are thought to be pathogenic and all are found in Europe. However, in North America, B. burgdorferi sensu stricto is the only identified pathogenic genospecies. Each of these three genospecies is associated with distinct clinical manifestations (Van Dam, A. P. et al., Clin. Infect. Dis. 17:708-717 (1993)). This implies that differences in genospecies may play an important role in the wide array of clinical manifestations observed in Lyme Disease.
OspA is a basic lipoprotein of approximately 31 kd, which is encoded on a large linear plasmid along with OspB, a basic lipoprotein of approximately 34 kd (Szczepanski, A., and J. L. Benach, Microbiol. Rev. 55:21 (1991)). Analysis of isolates of B. burgdorferi obtained from North America and Europe has demonstrated that OspA has antigenic variability, and that several distinct groups can be serologically and genotypically defined (Wilske, B., et al., World J. Microbiol. 7: 130 (1991)). Other Borrelia proteins demonstrate similar antigenic variability. Surprisingly, the immune response to these outer surface proteins tends to occur late in the disease, if at all (Craft, J. E. et al., J. Clin Invest. 78: 934-939 (1986); Dattwyler, R. J. and B. J. Luft, Rheum. Clin. North Am. 15: 727-734 (1989)). Furthermore, patients acutely and chronically infected with B. burgdorferi respond variably to the different antigens, including OspA, OspB, OspC, OspD, p39, p41 and p93.
As an infected tick begins to feed on a mammal, the synthesis of another outer surface protein, outer surface protein C or OspC, is induced (Schwan, T. G., et al, Proc. Natl. Acad. Sci. 2:2909-2913 (1995)). Thus, in early infection, OspC is the major outer membrane protein expressed by the spirochete (Fung, B. P., et al., Infect. Immun. 62:3213-3221 (1994); Padula, S. J., et al., J. Clin. Microbiol., 32:1733-1738 (1994)). Even through OspC has been demonstrated to have limited surface exposure (Cox, D. L., et al., Proc. Natl. Acad. Sci., 93:7973-7978 (1996); Mathiesen, M. M., et al., Infect. Immun. 66:4073-4079 (1998)), OspC is a potent immunogen. Immunization with OspC is protective against tick-transmitted Borrelia infection (Gilmore Jr., R. D., Infect. Immun. 64:2234-2239 (1999)). However, because OspC is highly variable in its sequence, the protection is limited to the Borrelia burgdorferi strain expressing the same allele of OspC. Challenge with heterologous isolates, expressing other ospC alleles results in infection (Probert, W. S., et al., J. Infect. D., 175:400-405 (1997)). OspC is a very diverse genetic locus (Jauris-Heipke, S., et al., Med. Microbiol. Immunol. 182:37-50 (1993)) as evidenced by the fact that Livey et al. found thirty-four alleles of OspC in seventy-six B. burgdorferi sensu lato isolates (Livey, I., et al., Mol. Microbiol. 18:257-269 (1995)).
Currently, Lyme Disease is treated with a range of antibiotics, e.g., tetracyclines, penicillin and cephalosporins. However, such treatment is not always successful in clearing the infection. Treatment is often delayed due to improper diagnosis with the deleterious effect that the infection proceeds to a chronic condition, where treatment with antibiotics is often not useful. One of the factors contributing to delayed treatment is the lack of effective diagnostic tools.
Vaccines against Lyme borreliosis have been attempted. Mice immunized with a recombinant form of OspA are protected from challenge with the same strain of B. burgdorferi from which the protein was obtained (Fikrig, E., et al., Science 250: 553-556 (1990)). Furthermore, passively transferred anti-OspA monoclonal antibodies (MAbs) have been shown to be protective in mice, and vaccination with a recombinant protein induced protective immunity against subsequent infection with the homologous strain of B. burgdorferi (Simon, M. M., et al., J. Infect. Dis. 164: 123 (1991)). In addition, there have been two independent trials of first generation vaccines for the prevention of Lyme disease that have studied the efficacy and safety of a vaccine based on recombinant outer surface protein A (OspA) (Sigal, L. H. et al., N. Engl. J. Med 339:216-222, 1998; Steere, A. C. et al., N. Engl. J. Med. 339:209-215, (1998)). However, a vaccine that consists of recombinant OspA may require frequent booster immunizations. An additional concern of OspA-based vaccines is the recent identification of a putative autoreactive OspA domain with a high degree of similarity to a region of human leukocyte function-associated antigen-1 (hLFA-1) (Gross, D. M. et al., Science, 281: 703-706 (1998)).
It has been noted that immunization with a single protein from a particular strain of Borrelia often does not confer resistance to that strain in all individuals (Fikrig, E. et al., J. Immunol. 7: 2256-1160 (1992)). There is considerable variation displayed in OspA, OspB and OspC, as well as p93, including the regions conferring antigenicity. Therefore, the degree and frequency of protection from vaccination with a protein from a single strain depend upon the response of the immune system to the particular variation, as well as the frequency of genetic variation in B. burgdorferi. In the case of vaccines directed against OspA, the vaccine is typically only effective against strains of Borrelia that express OspA that is homologous to OspA from which the vaccine was derived.
Another limitation of current OspA Lyme Disease vaccines is that they are directed against an antigen that is expressed predominantly in the tick vector. Indeed, recent reports have indicated that Borrelia burgdorferi in infected ticks alter their surface expression by increasing expression of OspC during ingestion of a blood meal (Schwan, T. G. et al., Proc. Natl. Acad. Sci. USA, 92: 2909-2913 (1995)). Thus, it seems that natural infection with B. burgdorferi does not elicit an antibody response to OspA, as it does against OspC.
Given the heterogeneity of antigenic determinants present in Borrelia proteins, a need exists for a vaccine and diagnostic tool which can provide immunogenicity to various strains and/or genospecies of Borrelia burgdorferi, as well as to more epitopes within a strain or genospecies. There is also a need for vaccines and diagnostic tools which detect antibody responses against immunoprotective targets that are expressed at different stages of the life cycle of Borrelia burgdorferi. This would allow for diagnosis and/or vaccination against all, or most forms, of Borrelia that cause systemic disease.